When assaying the binding of ligands to proteins it is often necessary to separate the ligand into proteinbound and unbound (free) fractions in order to determine quantities or perform measurements on specific fractions. The separation can be effected with ultrafiltration and gel filtration; selective precipitation of protein together with the bound ligand such as precipitation with ammonium sulfate, polyethylene glycol or a second antibody; selective adsorption of the free ligand by an adsorbent such as a suspension of activated charcoal or ion exchange resin; or a solid support phase on which the binding proteins or adsorbents are immobilized such as antibody coated tubes and resin sponges.
Various adsorption techniques and applications are demonstrated by the following references:
Herbert, U.S. Pat. No. 3,442,819 teaches that charcoal coated with a molecular sieve which will prevent large molecules of complexes from reaching and adhering to the carbon can be used to separate components in biochemical assays by adsorbing particular molecules. The selection of coating material permits the selective adsorption of one compound from another, i.e., generally molecules smaller than the pore size of the molecular sieve will pass through the sieve and be adsorbed, whereas, the molecules as large or larger than the sieve will not pass through the sieve and will remain in the assay medium.
Shannon, et al., U.S. Pat. No. 3,947,564 demonstrates the separation of thyroxine from serum proteins by the adsorption of thyroxine onto a montmorillonite clay in acid solution with subsequent centrifugation.
Lewin, et al., U.S. Pat. No. 3,937,799 illustrates the adsorption of unbound vitamin B-12 from an assay medium using a premeasured tablet of bentonite.
Applicant is also aware of a commercially available filtration device marked by Isolab which utilizes charcoal that has been bound to a polyethylene support. This adsorbent reagent is granular and amorphous. As a result, there is a variable amount of charcoal on each particle. Employing this reagent requires measuring by weight or volume to disperse the desired amount for each use. Usually, the reagent is packed in a column to be used as a filtration or chromatographic device. It is alleged that the advantage of the plastic support is that it enhances the flow rate of fluid through the column by preventing the charcoal from packing tightly.